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Am J Physiol Cell Physiol (June 11, 2003). doi:10.1152/ajpcell.00477.2002
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Submitted on October 11, 2002
Accepted on June 2, 2003

Functional Role of the NPxxY Motif in Internalization of the Type 2 Vasopressin Receptor in LLC-PK1 Cells

Richard Bouley1*, Tian-Xiao Sun1, Melissa Chenard1, Margaret McLaughlin1, Mary McKee1, Herbert Y Lin1, Dennis Brown1, and Dennis A Ausiello1

1 Program in Membrane Biology and Renal Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA

* To whom correspondence should be addressed. E-mail: bouley{at}receptor.mgh.harvard.edu.

Interaction of the type 2 vasopressin receptor (V2R) with hormone causes desensitization and internalization. To study the role of the V2R NPxxY motif (which is involved in the clathrin-mediated endocytosis of several other receptors) in this process, FLAG-tagged wild-type V2R and a Y325F mutant V2R were expressed in LLC-PK1a epithelial cells that have low levels of endogenous V2R. Both proteins had a similar apical (35%) and basolateral (65%) membrane distribution. Substitution of Tyr325 with Phe325 prevented ligand-induced internalization of V2R determined by [3H]-AVP binding and immunofluorescence, but did not prevent ligand binding or signal transduction via adenylyl cyclase. Desensitization and resensitization of the V2R-Y325F mutation occurred independently of internalization. The involvement of clathrin in V2R downregulation was also shown by immunogold electron microscopy. We conclude that the NPxxY motif of the V2R is critically involved in receptor down-regulation via clathrin-mediated internalization. However, this motif is not essential for the apical/basolateral sorting and polarized distribution of the V2R in LLC-PK1a cells, nor for adenylyl cyclase-mediated signal transduction.




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